Deacetylated polysaccharide S-60

ABSTRACT

Deacetylated Polysaccharide S-60, prepared by deacetylating the polysaccharide S-60 produced by fermentation of Pseudomonas elodea, ATCC 31461, has valuable properties in both the clarified and non-clarified form, and is useful as an agar substitute or a shapeable room deodorant. 
     The non-clarified deacetylated gum contains about 17% protein and principally carbohydrate, which comprises glucuronic acid (˜13% based on wt. gum) and the neutral sugars rhamnose and glucose in the approximate molar ratio 3:2. 
     The clarified, deacetylated gum contains mostly carbohydrate and no more than about 2% protein.

CROSS-REFERENCE

This is a continuation-in-part of copending U.S. Ser. No. 47,505, filedJune 8, 1979, now abandoned, which is a continuation-in-part of U.S.Ser. No. 966,538, filed Dec. 4, 1978, now abandoned.

Heteropolysaccharide S-60 is disclosed and claimed in a copendingapplication filed on even date herewith Ser. No. 178,054 which is acontinuation-in-part of U.S. Ser. No. 47,598, filed June 8, 1979, nowabandoned, which is a CIP of U.S. Ser. No. 966,531, filed Dec. 4, 1978,now abandoned.

BACKGROUND OF THE INVENTION

Compound S-60 may be prepared by fermentation of a suitable nutrientmedium with a hitherto undescribed organism. Based on extensivetaxonomic studies, the organism has been designated to be a new species,Pseudomonas elodea. An unrestricted permanent deposit of an organism ofthis species employed in making our heteropolysaccharide was made withthe American Type Culture Collection on Nov. 21, 1978, under AccessionNo. ATCC 31461.

Various classification keys for the genus Pseudomonas and the culturedescriptions of Pseudomonas species are found in the 7th Edition ofBergey's Manual (Breed et al., (1957)) and the 8th Edition of Bergey'sManual (Doudoroff et al., (1974), as well as by other schools in variouspublications; Hugh and Gilardi, 1974, Pseudomonas, Manual of ClinicalMicrobiology, 2nd ed., Lennette et al., Eds., pp. 250-269. AmericanSociety for Microbiology, Washington, D.C.; Weaver et al., 1972,Identification of Unusual Pathogenic Gram-Negative Bacteria, E. O. King,Center for Disease Control, Atlanta; Iizuka et al., 1963, Attempt atGrouping the Genus Pseudomonas, J. Gen. Appl. Microbiology 9:73-82; andHendric et al., 1966, Identification of Certain Pseudomonas Species,Identification Methods for Microbiologists, Part A, Gibbs et al., Eds.,pp. 1-7, Academic Press, New York.

These keys and descriptions were searched for a Pseudomonas specieshaving morphological and cultural characteristics similar to those ofATCC 31461. The following considerations make the assignment of a newPseudomonas species justified and necessary.

DESCRIPTION OF THE STRAIN 1. Characteristics of Cell Morphology

Single cells, straight or often curved, generally 0.6-0.8 by 2.0-3.0 μm,often with tapered end. The older cultures become larger and longer(0.8-1.0 by >3 μm), misshaped cells and pleomorphism appear, especiallyon media with limited amount of carbohydrates. On the contrary, cellskeep rather consistent rod shapes when grown on media withcarbohydrates, but again, most cells become large and pleomorphismdevelops during prolonged incubation. Gram-negative, non-capsulated,poly-β-hydroxybutyrate and polyphosphate granules are seen especially incultures of nitrogen-deficient media. Motile by polar multitrichousflagellation; one to four flagella are inserted at the polar end andoccasionally subpolar insertion may be seen.

2. Characteristics of Colonial Morphology

On nutrient agar plates, small (0.8-1.1 mm in diameter) and large(3.2-3.5 mm in diameter) colonies appear. They are yellow carotenoidpigmented, smooth, round, and convex to pulvinate. Large colonies oftenhave a concentric wrinkle. The surface of these colonies has a hard butnot viscid texture and entire colonies are removed if pushed by a loop.On YM agar plates, only one type of relatively large (˜6-7 mm indiameter) yellow, round, smooth, slimy and convex colonies appear. Slimyelastic membranes form on the surface of these colonies and wholesurface membranes (of colonies) can be removed. The secondary growth mayoccur around the edge of the original colonies. The color of thesecolonies is darker yellow towards the center than the edge andconcentric color formation appeared. In addition to the intracellularyellow carotenoid pigment(s), diffusible brown pigment developed as aresult of autooxidation after prolonged incubation. This phenomenon ismore easily recognized on Nutrient agar. No fluorescent pigment wasproduced.

3. Physicological and Biochemical Characteristics

The growth range of the strain S-60 is about 20° C. to 41° C. No growthoccurs at 4° C. 3.0% NaCl is sufficient to inhibit the growth and thestrain is capable of growth at pHs between 5 and 11.

Acid, but no gas is produced from almost all carbohydrates but not frompolyalcohols. Urease may be produced. MR, VP, and indole tests were allnegative. Argine dihydrolase, lysine and ornithine decarboxylase are notproduced. Acid and reduction occurs in litmus milk. Lipolytic egg yolkreaction is negative. Gelatin is weakly hydrolyzed but not casein,starch, alginate, pectin, cellulose, chitin and DNA.

4. Susceptibility to Antibiotics

The strain is very susceptible to Kanamycin, Neomycin,Chlortetracycline, Erythromycin, but not Streptomycin and Penicillin.

5. Nutritional Characteristics

Organic growth factors are not required and ammonium salts serve as solenitrogen source. At least 35 organic compounds were utilized, thosebeing most carbohydrates other than D-ribose, starch, 2-ketogluconate,and mucate. In addition, acetate, caproate, caprylate, pelargonate,succinate, azelate, L-malate, DL-β-hydroxybutyrate, pyruvate, ethanol,n-propanol, p-hydroxybenzoate, phenylacetate, L-α-alanine, L-threonine,L-leucine, DL-isoleucine, L-aspartate, L-glutamate, and L-tyrosine wereutilized.

6. The G+C Content of the DNA

Evaluation of the DNA resulted in the mole % to be ˜68 (by Tm).

                                      TABLE 1                                     __________________________________________________________________________    Biochemical and Other Miscellaneous Tests Employed                            for the Strain S-60                                                           __________________________________________________________________________    Oxidase:                                                                      Kovac's         + (weak)                                                                              Hydrolysis of:                                        Pathotech       + (weak)                                                                              Gelatin      + (weak)                                                         Casein       -                                        Catalase        +       Starch       -                                        OF medium:                                                                    Oxidative       +       Tween 80     +                                        Fermentative    -       Pectin       -                                        Gas from glucose                                                                              -       Alginate     -                                        H.sub.2 S production: TSI                                                                     -       Cellulose    -                                        from cystine    +       Chitin       -                                        Ammonium from peptone                                                                         +       DNA          -                                        β-Galactosidase (ONPG)                                                                   + (API) Esculin      +                                        Arginine dihydrolase                                                                          -                                                             Lysine decarboxylase                                                                          -       Growth on various media:                              Ornithine decarboxylase                                                                       -       EMB agar     -                                        Tryptophan deaminase                                                                          -       MacConkey agar                                                                             -                                        Phenylalanine deaminase                                                                       -       SS agar      -                                        Urease          +/±  Mannitol salt agar                                                                         -                                        Indole          -       TCBS agar    -                                        MR test         -       Tinsdale tellurite                                    VP test         -       blood agar   +                                        Nitrate reduction                                                                             -       Pseudosel agar                                                                             -                                        Nitrate reduction                                                                             -                                                             Denitrification -       Pigment production:                                   N.sub.2 -fixation:      King A medium                                                                              -                                        Growth in Burk's medium                                                                       +       King B medium                                                                              -                                        Nitrogenase activity                                                                          -                                                             Malonate (oxidation)                                                                          -       Dye reaction:                                         Phosphatase     +       Congo red    -                                        Haemolysis (sheep blood)                                                                      -       Nite blue    -                                        Litmus milk: acid, reduction only                                             3-ketolactose production                                                                      -                                                             Survival at 60° C. for 30 min.                                                         -                                                             TSI:                                                                          Slant           color no change                                               Butt            color no change                                               Gas             -                                                             Egg Yolk Reaction                                                                             -                                                             __________________________________________________________________________

FERMENTATION CONDITIONS

Heteropolysaccharide S-60 is produced during the aerobic fermentation ofsuitable aqueous nutrient media under controlled conditions via theinoculation with the organism of the Pseudomonas elodea species. Themedia are usual media, containing source of carbon, nitrogen andinorganic salts.

In general, carbohydrates (for example, glucose, fructose, maltose,sucrose, xylose, mannitol and the like) can be used either alone or incombination as sources of assimilable carbon in the nutrient medium. Theexact quantity of the carbohydrate source or sources utilized in themedium depend in part upon the other ingredients of the medium but, ingeneral, the amount of carbohydrate usually varies between about 2% and4% by weight of the medium. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium. In general, many proteinaceous materials may be used as nitrogensources in the fermentation process. Suitable nitrogen sources include,for example, yeast hydrolysates, primary yeast, soybean meal, cottonseedflour, hydrolysates of casein, corn steep liquor, distiller's solublesor tomato paste and the like. The sources of nitrogen, either alone orin combination, are used in amounts ranging from about 0.05% to 0.2% byweight of the aqueous medium.

Among the nutrient inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate,and like ions. Also included are trace metals such as cobalt, manganese,iron and magnesium.

It should be noted that the media described in the examples are merelyillustrative of the wide variety of media which may be employed, and arenot intended to be limitative.

The fermentation is carried out at temperatures ranging from about 25°C. to 35° C.; however, for optimum results it is preferable to conductthe fermentation at temperatures of from about 28° C. to 32° C. The pHof the nutrient media for growing the Pseudomonas culture and producingthe polysaccharide S-60 can vary from about 6 to 8.

Although the novel polysaccharide S-60 is produced by both surface andsubmerged culture, it is preferred to carry out the fermentation in thesubmerged state.

A small scale fermentation is conveniently carried out by inoculating asuitable nitrient medium with the culture and, after transfer to aproduction medium, permitting the fermentation to proceed at a constanttemperature of about 30° C. on a shaker for several days.

The fermentation is initiated in a sterilized flask of medium via one ormore stages of seed development. The nutrient medium for the seed stagemay be any suitable combination of carbon and nitrogen sources. The seedflask is shaken in a constant temperature chamber at about 30° C. for1-2 days, or until growth is satisfactory, and some of the resultinggrowth is used to inoculate either a second stage seed or the productionmedium. Intermediate stage seed flasks, when used, are developed inessentially the same manner; that is, part of the contents of the flaskfrom the last seed stage are used to inoculate the production medium.The inoculated flasks are shaken at a constant temperature for severaldays, and at the end of the incubation period the contents of the flasksare recovered by precipitation with a suitable alcohol such asisopropanol.

For large scale work, it is preferable to conduct the fermentation insuitable tanks provided with an agitator and a means of aerating thefermentation medium. According to this method, the nutrient medium ismade up in the tank and sterilized by heating at temperatures of up toabout 121° C. Upon cooling, the sterilized medium is inoculated with apreviously grown seed of the producing culture, and the fermentation ispermitted to proceed for a period of time as, for example, from 2 to 4days while agitating and/or aerating the nutrient medium and maintainingthe temperature at about 30° C. This method of producing the S-60 isparticularly suited for the preparation of large quantities.

S-60 is produced at a conversion efficiency of about 50%, resulting in avery high viscosity beer (4,000 to 8,000 cP). The product is recoveredfrom the fermentation medium by precipitation with a suitable alcohol,such as isopropanol.

HETEROPOLYSACCHARIDE S-60

The heteropolysaccharide produced by P. elodea is composed of about 50%carbohydrate and 50% insoluble material which contains protein (10 to15% based on total weight).

The carbohydrate portion contains 3 to 4.5% O-acetyl groups as theO-glysosidically linked ester, about 11% glucuronic acid (both based ontotal weight of the heteropolysaccharide) and the neutral sugarsrhamnose and glucose. The approximate molar ratio of rhamnose to glucoseis 3:2. The rhamnose and glucose are linked 1,4 and the negativespecific rotation ([α]₅₈₉ ²⁵ =-45%) of the deacetylated, clarifiedmaterial indicates that the majority of the glycose units are β-linked.The heteropolysaccharide is anionic.

The acetyl content of 4.5% was determined by treating a 0.2% aqueoussolution of S-60 gum with an alkaline, hydroxylamine reagent followed bytreatment with an acidic ferric chloride reagent [S. Hestrin (1949) J.Biol. Chem. 180 249-261].

The neutral sugars of polysaccharide S-60 were determined by dissolvingten mg. of the product in 2 ml 2N H₂ SO₄, and the mixture is heated at100° C. for 4 hours. The resulting solution is cooled, neutralized withbarium hydroxide and the pH is brought to 5-6 with solid carbon dioxide.The resulting precipitate of barium sulfate is removed by centrifugationand the supernatent is concentrated to a syrup under reduced pressure.The sugars in the hydrolysate are tentatively identifed by gasliquidchromatography of their aldononitril acetate derivatives on aHewlett-Packard Model 5750 chromatograph using 3% by weight OV-225 on80/100 mesh Gas Chrome Q at 210° C. The sugars are identified andquantitated by comparison with authentic standards [J. K. Baird, M. J.Holroyde, and D. C. Ellwood (1973) Carbohydr. Res. 27 464-467].

The various neutral sugars of the polysaccharides were alsocharacterized by use of descending paper chromatography on Whatman No. 1chromatography paper using as the solvent the upper layer of pyridine:ethyl acetate-water (2:5:5). Chromatograms were stained using silvernitrate dip and acid analine phthalate spray reagent. Component sugarswere identified by co-chromatography with sugar standards and by thespecific-color reaction with the aniline phthalate reagent.

The glycosidic linkages have been determined by methylation of thepurified polysaccharide followed by hydrolysis and quantitation of theO-methyl sugars as the alditol acetate derivatives. The derivatives wereseparated and identified by gas liquid chromatography--mass spectrometryusing a Hewlett-Packard Model 5992 GC/MS fitted with 1.5 ft. glasscolumns containing 3% OV-225 at 170° C. The derivatives of 2,3di-o-methylrhamnose and 2, 3, 6-tri-o-methylglucose were identified bycomparing the GC/MS spectra and relative retention times with those ofknown O-methyl sugar standards and with published data.

The uronic acid content of the polysaccharide was determined by twoseparate methods. In one method the sample was decarboxylated with 19%hydrochloric acid and the liberated carbon dioxide was trapped instandard sodium hydroxide and determined by back titration [B. L.Browning (1967) Methods of Wood Chemistry II, 632-633] and by thecarbazole colorimetric method [T. Bitter and H. M. Muir (1962) Anal.Biochem. 4 330-334].

Paper electrophoresis was used for the separation and tentativeidentification of the uronic acids present in the neutralized acidhydrolysate described above. Aliquots of this and known uronic acidstandards were applied to Camag electrophoresis paper No. 68-011 andelectrophoresis was carried out for 2.0 hours in a pH 2.7 buffer using aCamag Model HVE electrophoresis apparatus. Chromatograms were air driedand stained with silver nitrate dip reagent to locate the uronic acidsbeing separated. Two major and one minor uronic acid spots were found.One of the major spots migrated with the same mobility as glucuronicacid (R_(GlcA) =1.0) while the other major spot (R_(GlcA) =0.85) and theminor spot (R_(GlcA) =0.73) had lower mobility. Under these sameconditions the relative mobility of known uronic acids are:

    ______________________________________                                                       R.sub.m                                                        ______________________________________                                        Glucuronic acid  1.0                                                          Mannuronic acid  0.96                                                         Galacturonic acid                                                                              0.65                                                         Guluronic acid   0.63                                                         ______________________________________                                    

These secondary spots are interpreted as being uronic acid containingoligosaccharide, which is the result of incomplete hydrolysis. Thus allof the uronic acid is glucuronic.

An infrared spectrum of native S-60 was made on dried material in a KBrpellet. The heteropolysaccharide evidenced peaks at: 3400 cm⁻¹, 2950cm⁻¹, 1740 cm⁻¹, and 1620 cm⁻¹ indicating hydroxyl groups, methylenegroups, carbonyl groups and carboxylic acid salts.

S-60 is incompatible with methylene chloride dye, substantiallyinsoluble in N,N-dimethylformamide, and soluble in DMSO or formamide.

A sample of S-60 shows the following elemental analysis: N-2.00%,C-42.62%, H-5.80%.

S-60 is readily soluble in water and is characterized by high viscosityat low concentration. Typical viscosities are 40-80 cP at 0.1%concentration and 1000-2000 cP at 0.5% when measured on a Brookfield LVFviscometer, 60 rpm, at 25° C., (spindles 2 and 3, respectively). The gumalso has a high rheological yield point; a 1% gum solution has a workingyield value of 60 dynes/cm², defined by the shear stress at a shear rateof 0.01 sec⁻¹ as measured using a Wells-Brookfield cone and plateviscometer in the spring relaxation mode.

Changes of pH in the range 3-11 do not substantially affect theviscosity of 0.5% solution of S-60.

The effect of temperature on the solution viscosity of S-60 is bothcharacteristic and unusual. The viscosity of a 0.5% S-60 solution isstable in the range of 20°-70° C. and abruptly undergoes a reversibledecrease when the temperature is increased above 70° C. A mostsignificant property of S-60, both in its native state and afterdeacetylation is the formation of thermoreversible gels after heatingand cooling. Following heating in the presence of various cations,native S-60 forms elastic or soft gels upon cooling, deacetylated S-60produces firm, non-elastic or brittle gels. Cations especially useful inthe formation of gels with S-60 are those of sodium, potassium,magnesium, calcium, barium, aluminum, and chromium.

S-60 is useful as a thickening, suspending and stabilizing agent inaqueous systems; for example, as an additive to textile printing pastesor in formulating low drift aqueous herbicide compositions, saladdressings, thickened puddings, and adhesive compositions.

DEACETYLATED, NON-CLARIFIED S-60

When the dry polymer or the fermentation broth is heated at high pH(using e.g., sodium carbonate or sodium hydroxide to pH 10) and hightemperatures 90°-100° C. for 10 minutes to 45 minutes deacetylationreadily occurs. The resultant deacetylated polysaccharide S-60 formsfirm non-elastic or brittle gels, useful in many industrial and foodapplications.

The composition of the deacetylated S-60 is about the same as that ofnative S-60. It is composed of about 50% carbohydrate and about 50%insoluble material which contains about 17% protein (based on totalweight).

The carbohydrate portion contains about 0% acetyl, 11-13% glucuronicacid (based on total weight) and the neutral sugars rhamnose and glucosein the approximate molar ratio 3:2. The rhamnose and glucose are linked1,4 and the majority of the glycose units are β-linked.

An infrared spectrum of deacetylated S-60 made on dried material in aKBr pellet evidenced peaks at: 3400 cm⁻¹, 2950 cm⁻¹, 1740 cm⁻¹, 1650cm⁻¹, and 1610 cm⁻¹.

A sample of deacetylated S-60 shows the following elemental analysis:N-2.67%, C-41.89%, H-6.07%.

One use for the deacetylated gum results from the fact that as a rigid,brittle gel, it can be molded, and used as a rigid structure, whichafter treatment with a suitable solution such as fragrance, findsapplicability as a room deodorant or an air freshener, or the like. Thedeacetylated gum can also be used in gel electrophoresis, as a gellingagent for use in microtomes in electronmicroscopy. The native anddeacetylated gums can also be used as suspending agents for barium inradiology, confectionary products, and as impression materials in toolmaking, dentistry and criminology.

DEACETYLATED, CLARIFIED S-60

The composition of the clarified, deacetylated S-60 differs from theunclarified, deacetylated S-60 in that it is primarily carbohydrate, theremaining insoluble material containing no more than about 2% protein(based on total weight).

An infrared spectrum of clarified, deacetylated S-60 made on driedmaterial in a KBr pellet evidenced peaks at: 3400 cm⁻¹, 2950 cm⁻¹, and1600 cm⁻¹.

A sample of deacetylated, clarified S-60 shows the following elementalanalysis: N-0.42%, C-36.85%, H-5.62%. The sample has a specificrotation:

    [α].sub.589.sup.25 =-45° C.

The deacetylated clarified gum is especially useful as an agarsubstitute in microbiological culture media for various clinical andnon-clinical microorganisms using a wide variety of culture media. Theconcentration of deacetylated clarified gum necessary to replace agar isdependent upon the medium used, but is within the range of about 0.5 toabout 1.25% (weight volume). Growth characteristics of microorganismsare quite similar to those on standard agar-based media.

The following detailed examples illustrate representative aspects ofthis invention.

EXAMPLE 1 Fermentation Procedure for Producing Heteropolysaccharide S-60

A. Culture Maintenance

Pseudomonas elodea, ATCC 31461, grows quite well on NA or YM agar, whichare used routinely for culture maintenance. The incubation temperatureis 30° C. The organism produces a yellow-orange carotinoid pigment and abrown diffusable pigment by 2-5 day's incubation.

B. Seed Preparation

Flask seeds are prepared in YM broth incubated at 30° C. When inoculatedwith a fresh plate culture, the YM broth cultures give good growth andgum formation by 24 hours.

The fermentation seed medium is the same as final fermentor medium,using one-gallon fermentors as seed vessels.

C. Final Fermentor Medium

The sodium- and potassium-salt form of the gum are prepared in adifferent media; both are described below. The organism has a definiteK+ requirement which must be added to the sodium fermentation medium.(3% dextrose can also be used). 0.2% corn steep liquor is added in bothmedia.

    ______________________________________                                        Sodium Salt        Potassium Salt                                             ______________________________________                                        3.0% Glucose       3.0% Glucose                                               0.01% MgSO.sub.4 . 7H.sub.2 O                                                                    0.01% MgSO.sub.4 . 7H.sub.2 O                              0.09% NH.sub.4 NO.sub.3                                                                          0.09% NH.sub.4 NO.sub.3                                    0.05% Promosoy     0.05% Promosoy                                             (soy protein concen-                                                          trate)                                                                        1 ml/L HoLe salts  1 ml/L HoLe salts                                          1 ppm Fe++         1 ppm Fe++                                                 0.05% Na.sub.2 HPO.sub.4                                                                         0.05% K.sub.2 HPO.sub.4                                    10 ppm K+                                                                     pH control = NaOH  pH control = KOH                                           ______________________________________                                    

HoLe salts are a trace element solution containing, tartrate, magnesiummolybdate, CoCl₃, ZnCl₂, CuCl₂, boric acid, manganese chloride andferrous sulfate.

When a low calcium product is desired, either of the media above areused with deionized water. Fermentation is complete aq. 50 hours; beerviscosity usually is 5000-8000 cp.

D. Recovery

Because of the gelling nature of the product, good fiber formationusually does not occur with ambient precipitation. However, we havefound that by pasteurization at 90°-95° C. for 3 minutes (during whichthe thick beer heat-thins considerably) excellent fibers can be obtainedby precipitation of the beer using two volumes of 99% isopropanol pervolume of beer without cooling. Average yields of 1.5% gum are obtainedwith 3% glucose in the 20L and 70L fermentors.

E. Drying

The product is recovered and dried at 50°-55° C. for up to one hour in aforced-air tray dryer.

F. Product Quality

One-percent viscosities of the K+ salt are usually in the range of 3000cP and for the lowcalcium sodium salt, approximately 7000 cP.

EXAMPLE 2 Deacetylation and Clarification of the HeteropolysaccharideS-60

Clarification of the gum, while not necessarily for all uses, is ofvalue when the gum is used as an agar substitute. Clarification can beaccomplished before deacetylation (in the native state) or afterdeacetylation. Since deacetylation uses hot caustic, and clarificationis done hot, the two procedures are easily and conveniently combined.Both deaceylation and clarification can be done with the beer or the drypolymer. For deacetylation, if the beer is used, the pH is adjusted to10 with KOH, the solution heated to 90° C. for 15 minutes, the pHadjusted to pH 7 with dilute H₂ SO₄, then clarified.

The general procedure for both deacetyation and clarification follows:

A. A 2% solution of beer or gum is heated to 90° C.

B. The pH is adjusted to 10 with KOH.

C. The temperature of the beer or solution was maintained at 90°-95° C.for 15 minutes.

D. The pH is adjusted to 6-8 with dilute HCl or H₂ SO₄.

E. Ten gms/liter of Super Aid were added to the material to be filtered.

F. The material was filtered through a pressure filter unit (pre-heated)with approximately a 6 mm bed of Super Aid and approximately 20-30 psi,using a filter unit with an area of 136 cm².

G. The filtrate is precipitated with isopropanol immediately to preventgelation and the fibers dried at 50° C. for one hour or less.

When no deacetylation is necessary, the above procedure is followed,except that the pH is not raised; rather than holding at 90° C., thesolution is immediately filtered, and then recovered.

Clarification is typically done on the potassium form; KCl can be addedto a solution of previously made product as necessary.

EXAMPLE 3 DEACETYLATED, NON-CLARIFIED S-60

S-60 fermentation liquor is heated to 90° C. and the pH adjusted to 10by addition of 25% KOH. The temperature is maintained for 15 minutes,followed by neutralization with concentrated HCl. This liquor is thendrained from the fermentor while hot and recovered with 2 volumes of 99%IPA. The fibers of deacetylated S-60 are collected, dried at 55° C. forone hour in a forced air tray drier, and then milled to a powder.

EXAMPLE 4 CLARIFICATION OF DEACETYLATION S-60

Deacetylated heteropolysaccharide S-60 as prepared in Example 3 isreconstituted to a 1% concentration in deionized water using a Lightninmixer for one hour followed by mixing on an Arti-Barinko with heating to50° C. The solution is then centrifuged at 10,000 R.P.M. (GSA head) for20 minutes in a Sorvall RC2-B refrigerated centrifuge keeping thetemperature above 40° C. The supernatant is decanted off andsequentially filtered through Gelman type AN Hydrophilic Acropormembranes (293 mm) of porosities of 5μ, 3μ, 1.2μ and 0.8μ. The filtrateis added to approximately 3-4 volumes of 99% isopropanol, the fiberscollected and dried briefly in the forced air tray drier at 55° C.,followed by milling to a powder. The product is the deacetylated,clarified S-60 gum of this invention.

EXAMPLE 5 Heteropolysaccharide S-60 Gelling Characteristics

A compilation of data comparing the native gum and the deacetylated gum,both in the K+ form and in the Ca++ form, with carrageenan and agarfollows:

    ______________________________________                                                    Gel                                                               Type        Nature   Melts    Sets   Hysteresis                               ______________________________________                                        Native S-60 Very     65-70° C.                                                                       65-70° C.                                                                     None                                                 Elastic                                                           Deacetylated                                                                  S-60                                                                            K.sup.+ Gel                                                                             Brittle  90° C.                                                                          31-48° C.                                                                     45-60° C.                           Ca.sup.2+ Gel                                                                           Brittle  90° C.                                                                          45-50° C.                                                                     45-50° C.                         Kappa Carrageenan                                                                         Brittle  40-95° C.                                                                       25-75° C.                                                                     15-20° C.                         Agar*       Brittle  60-97° C.                                                                       32-39° C.                                                                     60° C.                            ______________________________________                                         *Bacteriological grade specs: gelling temperature range                       33°-39° C., melting temperature 70° C. minimum.          (Whistler's "Industrial Gums")                                           

    Minimum Gelling Concentration                                                 Kappa Carrageenan                                                                          0.3%                                                             Agar         0.04%                                                            Deacetylated S-60                                                                          0.05%                                                            (calcium gel)                                                             

Note above that there is a wide range of temperatures given for settingand melting of all the various types of gels. For agar, the variationsare primarily due to type of seaweed while the kappa carrageenan thepotassium ion concentration determines the gel characteristics. The gelsof deacetylated S-60 are primarily characterized by the degree ofdeacetylation. With only slight deacetylation the gels set at highertemperatures and are more elastic; in fact, a wide range of gel typesfrom very elastic to very brittle is possible, depending on the degreeof deacetylation. The gels appear to be more similar to agar than tokappa carageenan, primarily because of the large hysteresis betweensetting and melting temperatures. It should be emphasized that they aredifficult to melt and the gel-sol transition is difficult to observe. Onthe other hand, the gelling temperatures can be easily defined since thegels set sharply within a few degrees from incipient gelation to solidgel.

EXAMPLE 6 Agar Replacement Using Deacetylated Clarified S-60

Several different media are prepared as follows:

    ______________________________________                                        Nutrient Agar                                                                 (A)  0.8% Nutrient Broth (Difco)                                                   1.5% Agar (Difco)                                                        (B)  0.8% Nutrient Broth (Difco)                                                   0.2% KCl                                                                      0.9% S-60                                                                Trypticase Soy Agar                                                           (A)  2.75% Trypticase Soy Broth (BBL)                                              1.5% Agar (Difco)                                                        (B)  2.75% Trypticase Soy Broth (BBL)                                              0.2% KCl                                                                      0.9% S-60                                                                Potato Dexrose Agar                                                           (A)  2.4% Potato Dextrose Broth (Difco)                                            1.5% Agar                                                                (B)  2.4% Potato Dextrose Broth (Difco)                                            0.2% KCl                                                                      0.9% S-60                                                                YM Agar                                                                       (A)  2.1% YM Broth (Difco)                                                         1.5% Agar (Difco)                                                        (B)  2.1% YM Broth                                                                 0.2% KCl                                                                      0.9% S-60                                                                Brain Heart Infusion Agar                                                     (A)  3.7% BHI Broth (Difco)                                                        1.5% Agar                                                                (B)  3.7% BHI Broth (Difco)                                                        0.2% KCl                                                                      0.9% S-60                                                                Burk's Agar                                                                   (A)                    (B)                                                    0.0584% K.sub.2 HPO.sub.4                                                     0.0225% KH.sub.2 PO.sub.4                                                     0.0174% K.sub.2 SO.sub.4                                                      0.0164% MgCl.sub.2 . 6H.sub.2 O                                               0.0064% CaCl.sub.2 . 2H.sub.2 O                                                                       Same as (A)                                           0.0005% FeCl.sub.3 . 6H.sub.2 O                                               0.00002% Na.sub.2 MoO.sub.4 . 2H.sub.2 O                                      0.0116% NaCl                                                                  1.0% Glucose                                                                  1.5% Agar (Difco)      0.2% KCl                                                                      0.9% S-60                                              ______________________________________                                    

Deionized water was used for all media. The ingredients were combined(except for Burk's) and autoclaved for 15-20 minutes at 121° C. and 15psi, cooled to 55° C., and poured into sterile petri dishes. Theingredients for Burk's were combined, except for the glucose, which wasautoclaved separately, and added to the medium after autoclaving. Afterthese nutrient plates had solidified and allowed to incubate at ambienttemperature for 24 hours to check for sterility, they were streaked withthe following fourteen cultures:

Agromyces ramosus ATCC 25173

Arthrobacter globiformis ATCC 8010

Aureobasidium pullulans NRRL YB-3861

Azotobacter indicus var myxogenes strain S-7 ATCC 21423

Azotobacter vinelandii ATCC 9047

Beijerinckia lacticogenes ATCC 19361

Erwinia cartovora ATCC 8061

Escherichia coli strain EG-47

Klebsiella pneumoniae strain S-53

Nocardia salmonicolor ATCC 21243 S-60

Streptococcus faecalis

Trichoderma longbrachiatum ATCC 13631

Zoogloea ramigera ATCC 25935

The plates were incubated at 30° C. for 3-5 days and then examined forgrowth. Good growth was observed for all strains on media made with S-60and little difference in colonial morphology was noted between mediamade with S-60 instead of agar. These results indicate that S-60 is anexcellent replacement for agar in microbiological media. The gel pointfor all media containing S-60, except BHI medium and TSA, was 42° C. Thegel point for BHI agar and TSA was 52° C. Agar typically gels at 42°-44°C.

EXAMPLE 7 Preparation of Molded Scented Gels Using Deacetylated S-60

    ______________________________________                                        (A)    1.50%         Polysaccharide S-60                                             0.75%         Sodium Carbonate                                                0.025%        Methyl p-hydroxybenzoate                                        3.00%         Rose fragrance                                                  4.00%         Isopropanol                                                     2.00%         Ethylene glycol                                                 88.50%        Water                                                    ______________________________________                                    

The native polysaccharide S-60 is blended with sodium carbonate andpreservative and dissolved in water at 70° C. The solution is thenfurther heated to 90° C. and held at that temperature for ten minutes todeacetylate the polysaccharide. After cooling to 60° C., the fragrancedispersed in the solvents is added and the mixture placed in the usualair freshener plastic molds. When the mixture cools to 38° C., gelationoccurred and a firm, self-supporting gel with good fragrance releasingproperties results.

(B) A dry deacetylated polysaccharide S-60 is prepared from thefermentate beer by adjusting the pH to 10.0 with dilute sodium hydroxideand heating to 90° C. for fifteen minutes. The solution is neutralizedto pH 7 with dilute hydrochloric acid, precipitated in two volumes ofispopropanol, dried, and milled. A solid air freshener gel is preparedfrom the deacetylated product in the following manner: 3.0 grams of thedeacetylated polysaccharide S-60 are blended with 1.5 grams potassiumchloride and 0.15 grams methyl p-hydroxybenzoate preservative and addedto 177 ml water. The solution is heated to 90° C. to dissolve; cooled to60° C., and a blend of 6.0 grams peppermint oil fragrance, 8.0 gramsisopropanol, and 4.0 grams ethylene glycol is added. The solution isplaced in plastic molds and cooled to ambient temperature. A strong,brittle gel with heavy mint fragrance forms. The gel can be unmoldedeasily and retains its shape without sagging.

EXAMPLE 8 Comparison of Native, Deacetylated, and Clarified andDeacetylated S-60

Three samples of S-60 (native, deacetylaed, and deacetylated andclarified) are analyzed. The following data are obtained.

    ______________________________________                                                                     Deacet-                                                              Deacet-  ylated and                                                  Native   ylated   clarified                                        ______________________________________                                        Uronic acid (%)                                                                            11         13       22                                           Acetyl (%)   3.0        0        0                                            Neutral sugar                                                                 (% mol. ratio)                                                                Glucose      (40)       (40)     (40)                                         Rhamnose     (60)       (60)     (60)                                         Proteins     10         17       2                                            Ash          7.0        8.0      9.5                                          ______________________________________                                    

What is claimed is:
 1. Deacetylated heteropolysaccharide S-60, whichcomprises (a) about 50% (wt./wt.) insoluble material of which about 34%(wt./wt.) is protein, and (b) about 50% (wt./wt.) carbohydrate whichcontains about 22-26% (wt./wt.) glucuronic acid, 0% acetyl groups, andthe neutral sugars rhamnose and glucose in the approximate molar ratio3:2, said rhamnose and glucose sugars being primarily 1,4 β-linked, saidheteropolysaccharide being further characterized in that it is anionic,and forms brittle, thermoreversible gels.
 2. A process for preparing thecompound of claim 1 which comprises heating a 1-5% aqueous solution ofthe heteropolysaccharide S-60 at a pH of about 10, at a temperature of90°-100° C. for from 10 minutes to 45 minutes, and recovering theproduct thereby produced.
 3. Deacetylated heteropolysaccharide S-60produced by a process which comprises heating a 1-5% aqueous solution ofheteropolysaccharide S-60 at a pH of about 10, at a temperature of90°-100° C. for from 10 minutes to 45 minutes, and recovering theproduct thereby produced.
 4. Deacetylated, clarifiedheteropolysaccharide S-60, which comprises no more than about 2%(wt./wt.) protein and carbohydrate said carbohydrate containing about22-26% (wt./wt.) glucoronic acid, 0% acetyl groups, and the neutralsugars rhamnose and glucose in the approximate molar ratio 3:2, saidrhamnose and glucose sugars being primarily 1,4 β-linked, saidheteropolysaccharide being further characterized in that it is anionic,and forms brittle, thermoreversible gels.